Bacterial phospholipases are lipolytic enzymes involved in bacterial virulence as well as organophosphate recycling. They often share similar kinetic behavior and limited sequence and structural homology with mammalian phospholipases that are involved in signal transduction. An important aspect of these enzymes is that while they are in general water- soluble, they carry out their catalysis at an interface. In many cases interaction of the enzyme with the interface at a distinct site precedes substrate binding to the catalytic site. The proposed work seeks to understand on a molecular level the complex interfacial behavior of two water-soluble lipolytic enzymes: Bacillus cereus non-specific phospholipase C (PLC) and Streptomyces chromofuscus phospholipase D (PLD). NMR, fluorescence, CD, and site-directed mutagenesis techniques will be used in conjunction with kinetic analyses to investigate phospholipid substrate, activator, and inhibitor interactions with each enzyme. General questions to be addressed include (i) what is the molecular nature of phospholipid allosteric interfacial site and how is the allosteric interfacial site and how is the allosteric signal transferred to the active site? (Ii) given the architecture of the active site, can substrate specificity or chemical reactivity be altered in a systematic way? And (iii) what are the critical parameters for phospholipid binding to these enzymes, both from the point of view of phospholipid activator and enzyme active sites? The results of these studies should provide insights into the interfacial behavior and catalytic mechanisms for the PLC and PLD enzymes. A molecular picture will be developed for interfacial binding sites as well as a solid understanding of how products (that include lipid second messengers), and substrate physical characteristics after phospholipase activity.